1. Field of the Invention
The invention is directed to methods, apparatus and kits for in vivo gene transfer and therapy, in particular by direct in vivo electrotransfection (DIVE).
2. Description of the Background
Transfection is a very important and common technique that is routinely used in modern biomedical and genetic applications. Transfection refers to a method of introducing nucleic acid material into a target cell in a non-lethal manner. Once transfection is used to introduce a nucleic acid into a cell, the nucleic acid may direct synthesis of new RNA and/or new proteins. These RNA and/or proteins may provide new functionality for the cell or suppress the expression of (turn off) other genes.
One of the most important uses for transfection is in gene therapy. Gene therapy refers to the treatment of certain disorders, especially those caused by genetic anomalies or deficiencies, by introducing specific engineered genes into a patient""s cells (the host cells). The gene introduced into the cell can treat disorders by expressing a sequence that the host cells, because of its anomalies or deficiencies, cannot express. Thus, for example, the treatment for diabetes may involve transfecting the cells of a diabetic patient with a gene construct that either expresses insulin or induces the expression of insulin. Similarly, sickle cell anemia may be treated by suppressing the expression of the defective sickle cell gene and inducing the expression of normal hemoglobin gene.
Gene suppression may occur by transfecting a gene which encodes a suppressor protein or an antisense nucleic acid construct. A suppressor protein is any protein that reduces or eliminates the expression of another gene. An antisense nucleic acid is a nucleic acid that is complementary to an expressed gene. The complementary nucleic acid may hybridize to the sense RNA of the targeted gene to form non-functional double stranded RNAs which is not translated into protein. In any case, the expression (i.e., translation) of the targeted gene is suppressed by expression (i.e., transcription) of antisense DNA.
Numerous techniques have been developed for transfection of cells in vitro. The basic goal of all transfection techniques is to introduce the nucleic acid into a target cell. Transfection techniques may be broadly classified as either direct or indirect methods. Direct methods involve the manual introduction of nucleic acid. Examples of direct transfection include microinjection or microprojectile transfection. Indirect transfection techniques are numerous but can be broadly classified into viral transfection techniques, liposome transfection techniques and phagocytosis techniques. Successful demonstration of these techniques in vitro has not necessarily been followed by success in vivo.
Direct transfection can be performed by microinjection, microprojectile transfection or by electrotransfection or laser transfection. Microinjection involves the manual injection of nucleic acid solutions into a cell by the use of a small needle (usually a drawn glass capillary) under a microscope. Microprojectile transfection involves the coating small particles with nucleic acid and shooting the particles into a cell with a high velocity gun. Laser transfection or electrotransfection involves puncturing a temporary hole in the cell membrane and allowing nucleic acid in the surrounding media to enter.
Direct transfection is labor intensive. A skilled operator can inject at most, between 200 and 500 cells per day in vitro. Direct transfection by micro injection is difficult or impossible in vivo due to the instability and vibrations of a living subject. Further, the number of cells that can be transfected per day is too small for a significant difference in a living subject. Likewise, microprojectile transfection, a procedure often involving gunpowder and high pressure air, are not practical for use on a patient.
Another disadvantage of current direct transfection techniques is that the procedures are only effective on cells and body parts that can be exposed and accessible to the microinjection needle or microprojectile gun. Thus, the interior of organs such as kidney, brain, bladder, lung and heart cannot be transfected without surgery and concomitant damage to these organs. Laser and electrotransfecting techniques have also not been found to be readily applied to living patients.
The major disadvantage of indirect in vivo transfection is low efficiency. One type of indirect transfection uses viruses to introduce nucleic acid into cells. The viruses most often used include SV40, polyoma, adenovirus, Epstein-Barr, vaccinia, herpes simplex, and retrovirus for mammalian cells and baculovirus, tobacco mosaic virus, cucumber mosaic virus, brome mosaic virus for non-mammalian cells. All viruses currently used in vivo suffer from low transfection efficiency.
An additional disadvantage of viral mediated transfection is the danger of using an infectious agent in a patient. In principle each of these viral techniques may be performed in a way that prevents transmission of infectious virus to the patient. In practice, each technique requires viral recombination in laboratories where inattentive or incompetent personnel may greatly increase the chances for an infectious virus contamination. The use of viruses involves significant risks because some viruses are potent pathogens in their wildtype state and other viruses carry oncogenes in their genomes.
Other potential disadvantages of viral vectors include the limited ability to mediate in vivo (as opposed to it vitro or ex vivo) transfection; the inability of retroviruses to infect non-dividing cells; possible recombination events in replication defective retroviral vectors resulting in infectious retrovirus; possible activation of oncogene or suppression of anti-oncogene due to random insertion, size limitations (less than 15 kb of DNA can be packaged in a retrovirus vector); and the potential immunogenicity of the viral vectors leading to an immune response.
Other indirect transfection methods such as liposome transfection and DNA-calcium phosphate transfection also suffer from low transfection efficiency in vivo. Further, these methods use solutions that may be incompatible with cell survival. In vivo, cell death, which may lead to organ failure, is a significant disadvantage. However, efforts to reduce cell death, such as the rapid introduction and removal of transfection solutions, also reduce transfection efficiency.
Clearly, there is a need for a new in vivo transfection method that can improve the efficiency of target cell transfection without the adverse side effects of current methods. Further, new transfection methods are needed to transfect target cells, such as those in the interior of organs, that are not normally accessible.
The present invention overcomes many of the limitations, problems and disadvantages associated with current strategies and designs for direct in vivo electrotransfection and provides apparatus, transfection kits, and methods for the direct in vivo electrotransfection (DIVE) transfection of tissues.
One embodiment of the invention is directed to a method for direct in vivo electrotransfection of a plurality of cells of a target tissue with a nucleic acid construct. The target is perfused with a transfection solution comprising a nucleic acid construct. At least a portion of the target tissue is surrounded with an exterior electrode. One or more interior electrode is placed within the target tissue. The perfusion and external and internal electrode may be performed or positioned in any order. An electric waveform is applied through the exterior electrode to transfect the target tissue.
One advantage of the method is the use of a large substantially planar surface as an exterior electrode. The exterior electrode can be wrapped around an organ or wrapped around a body. In contrast to needle or multiple needle electrodes which provide an uneven and localized electric field, the large planar surface electrode can provide a more uniformed electric field which would, in turn, lead to more uniform transfection. Planar means sheet-like. Thus a planar electrode may not be flat, but may be wrapped around an organ like a bandage or a bed sheet.
The transfection solution may be any electrotransfection solution such as physiological saline and/or phosphate buffered saline. The salt content of the transfection solution may be increased or decreased to change the effective propagation of the electric field. This change and adjustment in salt content is particularly useful in a hollow organ, such as a bladder, which is filled with the transfection solution during DIVE.
Another embodiment of the invention is directed to a method for selectively transfecting a subsegment of the cells of a target tissue, which can be an organ, using direct in vivo electrotransfection. In the method, a subsegment of the target tissue is perfused with a transfection solution comprising a nucleic acid construct. An exterior electrode is positioned to surround at least a portion of said target tissue. One or more interior electrodes is placed within the target tissue. Then an electric waveform is applied through the exterior electrode and the interior electrode to cause the transfection of a subsegment of the target tissue. Transfection specificity is maintained because only cells in contact with the transfection solution are transfected. The exterior electrode may be positioned on the skin of the patient if the electric conduction is sufficient. Electric conduction may be facilitated by the application of a electroconductive gel between the exterior electrode and the skin. This method may be useful, for example, if it is only desired to transfect a subsegment of an organ. For example, the bladder lining may be selectively transfected.
Other embodiments and advantages of the invention are set forth, in part, in the description which follows and, in part, will be obvious from this description and may be learned from the practice of the invention.